Iron complexes of hydroxypyridones useful for treating iron overload

ABSTRACT

Compounds in which two or more rings, being a 3-hydroxpypyrid-2-one, 3-hydroxypyrid-4-one or 1-hydroxpyrid-2-one, are linked are of value in the treatment of patients having a toxic concentration of a metal, particularly iron, in the body while the iron complexes of such compounds are of value in the treatment of iron deficiency anaemia.

This application is a continuation of application Ser. No. 06/944,355,filed Dec. 22, 1986, now abandoned, which is a division of applicationSer. No. 06/651,684, filed Sept. 18, 1984, now U.S. Pat. No. 4,666,927.

This invention relates to compounds for use in pharmaceuticalcompositions.

BACKGROUND OF THE INVENTION

Certain pathological conditions such as thalassaemia, sickle cellanaemia, idiopathic haemochromatosis and aplastic anaemia are treated byregular blood transfusions. It is commonly found that such transfusionslead to a widespread iron overload, which condition can also arisethrough increased iron absorption by the body in certain othercircumstances. Iron overload is most undesirable since, followingsaturation of the ferritin and transferrin in the body, deposition ofiron can occur and many tissues can be adversely affected, particulartoxic effects being degenerative changes in the myocardium, liver andendocrine organs. Such iron overload is most often treated by the use ofdesferrioxamine. However, this compound is an expensive natural productobtained by the culture of Streptomyces and, as it is susceptible toacid hydrolysis, it cannot be given orally to the patient and has to begiven by a parenteral route. Since relatively large amounts ofdesferrioxamine may be required daily over an extended period, thesedisadvantages are particularly relevant and an extensive amount ofresearch has been directed towards the development of alternative drugs.However, work has been concentrated on three major classes of ironchelating agents or siderophores, namely hydroxamates, ethylenediaminetetra-acetic acid (EDTA) analogues and catechols. The hydroxamatesgenerally suffer from the same defects as desferrioxamine, beingexpensive and acid labile, whilst the other two classes are ineffectiveat removing iron from intracellular sites. Moreover, some cathecholderivatives are retained by the liver and spleen and EDTA analoguespossess a high affinity for calcium and so are also likely to haveassociated toxicity problems.

We have accordingly studied the iron chelating ability of a wide rangeof compounds and in UK patent applications 8308056 (published as GB2118176A), 8407181 (published as GB 2136807A), as well as in thecorresponding U.S. applications, application Ser. No. 478,493 (U.S. Pat.No. 4,840,958), application Ser. No. 592,271 (U.S. Pat. No. 4,585,780)and application Ser. No. 651,772 (U.S. Pat. No. 4,587,240; reissueapplication Ser. No. 253,579, we describe certain compounds which wehave identified as being of particular use for the treatment ofconditions involving iron overload. These compounds consist of3-hydroxypyrid-2-ones and 3-hydroxypyrid-4-ones having a substituent onthe nitrogen atom and optionally also on one or more of the carbon atomsof the ring, and 1-hydroxypyrid-2-ones which are substituted on one ormore of the carbon atoms of the ring. We have now found that compoundscontaining two or more linked 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-oneor 1-hydroxypyrid-2-one rings are of significant value in the treatmentof iron overload.

DESCRIPTION OF THE INVENTION

Accordingly the present invention comprises a compound in which two ormore rings, being a 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one or1-hydroxypyrid-2-one, are linked.

The hydroxypyridone rings in compounds according to the presentinvention may be identical or may differ, either varying between thethree forms of hydroxypyridone and/or, since the ring may besubstituted, between differing rings of the same basic form. In general,however, there is little advantage to be gained from the presence ofdissimilar rings and this can complicate the synthesis of the compoundso that it is preferred that each hydroxypyridone ring in the compoundis of the same one of the three forms and conveniently that each ringpossesses the same substitution or lack of substitution.

Although compounds according to the present invention may containvarying numbers of rings, for example up to 10 or even 100, compoundscontaining 2, 3 or 4 rings are of particular interest with the 3 ringcompounds being of especial use in the treatment of conditions involvingiron overload.

The ability of both the free compound and its iron complex to permeatemembranes is important in the context of the treatment of iron overload,and it is also desirable for both to possess some degree of watersolubility. A good indication of the physical properties of a compoundand its iron complex in this respect is provided by the value of thepartition coefficient (K_(part)) obtained on partition between n-octanoland tris hydrochloride (20 mM, pH 7.4; tris representing2-amino-2-hydroxymethylpropane 1,3-diol) at 20° C. and expressed as theratio (concentration of compound in organic phase)/(concentration ofcompound in aqueous phase). Where the compounds are to be employed in acontext requiring translocation across membranes, then preferredcompounds show a value of K_(part) for the free compound of above 0.02but less than 3.0, especially of above 0.2 but less than 1.0, togetherwith a value of K_(part) for the smallest neutral iron(III) complex inwhich there is an internal balance of charges of above 0.02 but lessthan 6.0, especially of above 0.2 but less than 1.0. [In a neutralcomplex there is an internal balance of charges between the iron cationor cations and the ligand or ligands covalently bound thereto withoutthe necessity for the presence of a non-covalently bound ion or ions toachieve balance. In the case of two ring compound, for example, thissmallest neutral iron(III) complex will contain 3 molar proportions ofthe compound:2 molar proportions of iron(III) but for the preferred ironbinding compounds containing three rings the complex will contain a 1:1molar ratio of compound: iron(III).] In other contexts, such as theintraveneous use of the compounds in vivo, the value of K_(part) is lesscritical. The comments which follow later upon preferred forms oflinking groups are in part directed towards providing compounds havingpartition coefficients in the free and complexed state which lie in thepreferred ranges indicated above, which preferences as to linking groupsalso apply broadly to compounds for use in the removal of the othermetals from the body as discussed hereinafter although the preferrednumber of rings present in the compound may then be different.

The hydroxypyridone rings may, for example, be substituted as discussedin the aforementioned UK patent applications for the single ringcompounds, allowing for the necessity to provide a point of linkage forthe various rings so that, for example, if the rings are linked throughthe nitrogen atoms thereof then this position of the ring may not beotherwise substituted.

Examples of types of substitution additional to the linking groups thusinclude, for 3-hydroxypyrid-2-one and 3-hydroxypyrid-4-one rings,replacement of the hydrogen atom attached to the nitrogen atom by analiphatic acyl group, by an aliphatic hydrocarbon group, or by analiphatic hydrocarbon group substituted by one or, except in the case ofionisable groups, more than one substituent selected from aliphaticacyl, alkoxy, aliphatic amine, aliphatic amide, carboxy, aliphaticester, halogen, hydroxy and sulpho groups and/or replacement of one ormore of the hydrogen atoms attached to ring carbon atoms by one of saidsubstituents, by an aliphatic hydrocarbon group, or by an aliphatichydrocarbon group substituted by an alkoxy, aliphatic ester, halogen orhydroxy group; and for 1-hydroxypyrid-2-one rings, replacement of one ormore of the hydrogen atoms attached to ring carbon atoms by asubstituent selected from aliphatic acyl, aliphatic amide, aliphaticamine, carboxy, cyano, aliphatic ester, halogen, hydroxy and sulphogroups, alkoxy groups and alkoxy groups substituted by an alkoxy,aliphatic amide, aliphatic amine, aliphatic ester, halogen or hydroxygroup, aliphatic hydrocarbon groups and aliphatic hydrocarbon groupssubstituted by an alkoxy, aliphatic ester, halogen or hydroxy group.

Further description of such substituents as are listed above is to befound in the earlier applications referred to hereinbefore but it may bementioned that the ionisable substituent groups are generally of lesserinterest than the others and, indeed, substitution of the rings otherthan for the purpose of the linkage thereof is of much less interestthan in the case of the single ring compounds. Since, as will bediscussed in more detail hereinafter, the 3-hydroxypyrid-2- and 4-onerings are preferably linked through the nitrogen atoms thereof and thehydroxy group on the nitrogen atoms of the 1-hydroxypyrid-2-ones isrequired for iron binding, such substitution as is present is moreusually limited to the carbon atoms of the ring and is preferablylimited to aliphatic hydrocarbon substituent groups. Moreover, althoughmore than one of the ring carbon atoms may be substituted, for exampletwo of such atoms, either by the same or different substituent groups,compounds in which only one, or none, of the ring carbon atoms aresubstituted are preferred. Substitution may occur at various positionsof the ring but, particularly when the ring carbon atoms are substitutedby the larger groups, there may be an advantage in avoiding substitutionon a carbon atom alpha to the ##STR1## system. These systems areinvolved in the complexing of the compound with iron and other metalsand the close proximity of one of the larger groups may lead to stericeffects which inhibit complex formation.

Where a ring is substituted by an aliphatic hydrocarbon group, thisgroup may be cyclic or acyclic, having a branched chain or especially astraight chain in the latter case, and may be unsaturated or especiallysaturated. Groups of from 1 to 6 carbon atoms, particularly of 1 to 4and especially of 1 to 3 carbon atoms, are of most interest. Alkylgroups are preferred, for example cyclic groups such as cyclopropyl andespecially cyclohexyl but, more particularly preferred are acyclicgroups such as methyl, ethyl, n-propyl and isopropyl. The main interestin aliphatic hydrocarbon group substituents in the context of thepresent invention lies, however, in the possibility of using single ringcompounds containing such a substituent on one or more carbon atomsthereof as being a more readily available starting material than thecorresponding unsubstituted compound for the preparation of themulti-ring compounds. In this respect, methyl substituent groups are ofparticular interest and have the advantage of not being so large as tocreate the problems referred to above. Thus, with the3-hydroxypyride-4-ones, where the use of methyl substituted compounds asstarting materials is of most relevance, it is likely, for reasons ofavailability, that any methyl substituent will in fact be located at the2- or 6-position.

Preferred ring systems present in the compounds of the present inventionare shown below, the free valency in each case indicating the preferredpoint of attachment of the linking group between one ring and another(it will be appreciated that the 1-hydroxypyrid-2-ones are tautomericcompounds, being alteratively named as 2-hydroxypyridine-1-oxides).##STR2##

The hydroxypyridone rings may be linked through various types of linkinggroup, the important feature of the compounds being the rings containedtherein and the nature of the linking groups therefore being of lesserimportance. From the point of view of ease of synthesis andnon-interference in the metal complexing action of the compounds,however, it is preferred to use linking groups which are either whollyof a hydrocarbon nature or which additionally contain one or more of thesame or different groups such as ##STR3## etc.

For reasons of simplification of the description only, the preparationof compounds according to the present invention will be furtherdescribed with particular reference to the preparation of two ring andthree ring compounds.

Examples of suitable linking hydrocarbon linking groups are a benzenegroup substituted by two or more methylene groups, and various aliphatichydrocarbon groups. The latter may conveniently contain between 6 and 18to 24 carbon atoms, especially between 6 or 8 and 12 carbon atoms, andwhen used in two ring compounds, where they are particularly useful, arepreferably straight chain alkylene groups, although when used incompounds containing more than two rings they must necessarily bebranched. Specific examples of such straight chain alkylene groups aregroups --(CH₂)_(n) -- in which n is an integer from 6 to 12, for example8, 9 or 10.

Alternative linking groups of especial interest are groups correspondingto a hydrocarbon group in which one or more carbon atoms are replaced bya nitrogen atom and/or in which one or more pairs of adjacent carbonatoms are replaced by a --CONH-- group. Such groups are preferably ofsimilar overall size (i.e. contain a similar total number of atoms inthe backbone or chain thereof) to the hydrocarbon groups describedabove. Certain of such groups are of particular interest in the contextof divalent linking groups for use in two ring compounds. These divalentgroups may conveniently correspond to a straight chain alkylene group inwhich one carbon atom is replaced by a --NH-- group and/or in which twopairs of adjacent carbon atoms are replaced by a --CONH-- group. Theywill often contain one imino group in a substantially central positionin the chain and/or one amido group located adjacent to each end of thechain, the carbonyl group thereof often being bonded to the terminal orpenultimate carbon atom of the chain. Specific examples of such linkinggroups are ##STR4## wherein a and b are each an integer from 2 to 6, forexample 2 to 5, and especially a=2, b=3, or a=3, b=4, or a=5, b=5; c isan integer from 1 to 5, especially 1 to 3, for example 1 or 2, and d isan integer from 2 to 8, for example c=1 and d=6, or c=2 and d=2 or 4;and e and f are each an integer from 2 to 4, for example c=1, e=2 andf=3, or c=2, e=2 and f=2 (values of d, e and f of 1 lead to linkinggroups containing the somewhat unstable ##STR5## grouping). As regardsthe overall size of these linking groups, in the case of (2) it ispreferred that 4≦2 c+d≦12 and in the case of (3) it is preferred that6≦2 c+e+f≦12. In the case of the 1-hydroxy-pyrid-2-ones, whereattachment of the linking group is likely to be to a carbon rather thana nitrogen atom, the linking groups used may often correspond to thosedescribed above but with an additional oxy group at each terminusthereof.

Trivalent linking groups of use in the present invention in thepreparation of three ring compounds may include both the hydrocarbon andmodified hydrocarbon types referred to hereinbefore. They are oftensimilar to the divalent linking groups described above, modified so asto provide three groups terminating in a free valency arranged about thecentre of the linking group instead of two. Groups of especial interestare a benzene group which is 1,3,5-substituted by three methylene groupsand tripod groups consisting of a nitrogen atom substituted by threealkylene groups, for example straight chain groups of 1 to 6 carbonatoms and particularly 4 or 5 carbon atoms, or similar substitutedbenzene groups or tripod groups in which the methylene or alkylenegroups are terminally substituted by a group --NHCO--(CH₂)_(c) --wherein c is as defined hereinbefore. In this latter case involvingfurther terminal substitution, the three alkylene groups attached to thenitrogen atom of the tripod group may generally be somewhat shorter thanpreviously indicated, preferably being straight chain and convenientlybeing of 1 to 4 carbon atoms, particularly of 2 or 3 carbon atoms when cis 1, for example of 2 carbon atoms each or of 2, 3 and 3 carbon atoms,respectively. Also of some considerable interest are trivalent groupssimilar to those divalent groups described above corresponding to analkylene group in which one carbon atom is replaced by a --NH-- groupand also two pairs of carbon atoms are replaced by a --CONH-- group. Inthis case, the trivalent group may be viewed as corresponding to abranched, trivalent hydrocarbon group in which a ##STR6## group isreplaced by ##STR7## and three pairs of adjacent carbon atoms arereplaced by a --CONH-- group. Alternatively, they may be viewed in acloser analogy to the divalent groups as corresponding to a straightchain alkylene group in which a --CH₂ -- group is respaced by ##STR8##group wherein g is an integer from 1 to 3, especially being 1, and twopairs of carbon atoms are replaced by a --CONH-- group. Again, thesegroups will often contain a nitrogen atom in a substantially centralposition in the chain and/or one amido group located adjacent to eachend of the chain, the carbonyl group thereof often being bonded to theterminal or penultimate carbon atom of the chain. Specific examples ofsuch linking groups are ##STR9## wherein c, e, f and g are as definedabove and h is an integer from 1 to 4, and especially, for (4), c=1,e=2, f=3 and g=1, or c=1, e=3, f=4 and g=1, or c=1, e=3, f=4 and g=2,and for (5), c=1 and e=f=h=2 or c=2 and e=f=h=2. As regards the overallsize of these linking groups, in the case of (4) it is preferred that6≦2 c+e+f≦12 whilst g is 1 to 3, and in the case of (5) it is preferredthat for each of 2 c+e+f, 2 c+e+h and 2 c+f+h the sum is greater than orequal to 6 but less than or equal to 12.

As regards tetravalent linking groups these may with particularconvenience contain a fully branched carbon atom ##STR10## which may beattached to four groups which are either wholly of a hydrocarbon natureor which additionally contain other groups such as those describedhereinbefore. An example of this type of linking group has the form##STR11## where each of a, a', b and b' is an integer from 1 to 6, forexample the same integer, a particular linking group having each of a,a', b and b'=1.

By way of further guidance in the selection of suitable linking groupsit may be stated that, for particularly preferred compounds, in the caseof the 3-hydroxypyrid-2-ones the nitrogen atoms of the rings mayconveniently be separated by 6 to 12, preferably 8 to 10 atoms, whilstwith the 3-hydroxypyrid-4-ones a convenient range is 8 to 16, preferably9 to 11 or 12 atoms. The situation with the 1-hydroxypyrid-2-ones willdepend on the position of attachment of the linking groups but, in thepreferred case of linking of the rings through the 4-position, thesituation is analogous to that of the 3-hydroxypyrid-4-ones.

The compounds according to the present invention may be synthesisedusing various different approaches. In the case of the3-hydroxypyridones, as mentioned hereinbefore, linkage is usuallyeffected through the nitrogen atom of the ring. One approach, which isparticularly applicable to the 3-hydroxypyrid-2-ones, involves reactionof the 3-hydroxypyridone, in a suitable proportion, with a reagentproviding the linking group which contains the appropriate number offunctional groups capable of reaction with the --NH-- group of thepyridone. This procedure is of particular interest for the preparationof two ring compounds and suitable functional groups include the iodogroup. Routes to C-substituted 3-hydroxypyridones are discussed in theUK patent applications 8308056 and 8407181 and their equivalentsreferred to hereinbefore but, as indicated before, the more readilyavailable compounds in which the carbon atoms of the ring areunsubstituted or substituted only by an aliphatic hydrocarbon group areof greater interest as starting materials.

A second approach involves reaction of a 3-hydroxypyridone which isN-substituted by a substituent containing a suitable first functionalgroup with a reagent containing suitable second functional groups forreaction with said first group, the linking group deriving from both theN-substituent and the reagent. The procedure is of particularapplication for both the 3-hydroxypyrid-2- and -4-ones in thepreparation of two ring and three ring compounds, and suitablefunctional groups include a combination of a carboxy group and an aminogroup, either one of which may be attached to the 3-hydroxypyridone andthe other of which to the reagent. Thus, the hydroxypyridone may, forexample, be N-substituted by a group --(CH₂)_(i) --CO₂ H or --(CH₂)_(i)--NH₂ wherein i is an integer from 1 to 5, for example 1, 2 or 3. Thereagent in the former case, which is the preferred one, may be a di- ortri-amine, etc., and in the later case a di- or tri-carboxylic acid,although it will be appreciated that it is usual, whether the carboxygroup is on the hydroxypyridone or the reagent, to activate it beforereaction. Such activation may, for example, involve the use of anactivated ester such as the p-nitrophenyl or 1-succinimyl ester and itwill be usual to protect the ring hydroxy group, for example bybenzylation, to prevent reaction thereof with the activated ester groupor other form of activated carboxy group.

Yet a third approach may be used in the case of the3-hydroxypyrid-4-ones where it is possible to react a di- or tri-amine,with a protected 3-hydroxy-4-pyrone, for example a 3-benzyloxy-4-pyrone,to replace the ring oxygen atom by a nitrogen atom of the reagent andthus effect linkage of two, three or more rings. Certain3-hydroxy-4-pyrones, such as the unsubstituted compound and the 2-methylsubstituted compound, maltol, are available commercially. Other readilyavailable compound include the 6-methyl substituted compound, isomaltol,and other 2-alkyl substituted compounds whose synthesis is described inU.S. application Ser. No. 310,141 (series of 1960).

A specific example of such a first approach as described above involvesthe use of a reagent I-(CH₂)_(n) -I, wherein n is as definedhereinbefore, and the reagent --C--(CH₂ Br)₄, to prepare compoundscontaining two or four linked 3-hydroxypyrid-2-one or3-hydroxypyrid-4-one rings, respectively, for example the rings (I) and(III) as shown hereinbefore. Specific examples of the second approachutilise a reagent H₂ N--(CH₂)_(d) --NH₂, H₂ N--(CH₂)_(e) --NH--(CH₂)_(f)--NH₂, ##STR12## where d is as defined hereinbefore, for example being2, 4, 6 or 8; e and f are as defined hereinbefore, for example being 2and 3 respectively or each being 2; and i, j and k are each an integerfrom 1 to 4, for example each being 2 or being 2, 3 and 3 respectively.Such reagents are used to prepare compounds containing two or threelinked rings by reaction with a 3-hydroxypyrid-2- or -4-one, for exampleone of the compounds (I) to (IV) as shown hereinbefore, which isN-substituted, for example by a group --CH₂ CO₂ H, --(CH₂)₂ --CO₂ H,--(CH₂)₃ --CO₂ H, --(CH₂)₄ --CO₂ H or --(CH₂)₅ --CO₂ H in activatedform. The third approach may be used with a reagent similar to thosedescribed for use in the second approach, for example a reagent H₂N--(CH₂).sub. a --NH--(CH₂)_(b) --NH₂ where a and b are as definedhereinbefore, or H₂ N--(CH₂)_(n) --NH₂ where n is as definedhereinbefore, for example being 8 or 9, or more conveniently with areagent corresponding to a linking group (2), (3) or (4) as describedabove having c=1 and terminating at the free valencies in an aminogroup, such reagents being used to prepare compound containing two orthree linked rings by reaction with 3-hydroxy-4-pyrones having aprotected hydroxy group, for example the O-benzylated pyronescorresponding to the pyridones (II), (III) and (IV), followed bydeprotection. (Such specific reagents may also be used in the secondapproach, although less conveniently, as an alternative to thosedescribed above.)

Consideration of these various approaches will show that, in someinstances, certain of the linking groups described above are preferredin the case of certain types of hydroxypyridone and also that, in someinstances, the same type of linking group may be produced by differentapproaches so that, for example, an amido function in the linking groupmay be present in the reagent used to form that group or may be producedby the reaction of an amine group and an activated ester group, one ofwhich is attached to the reagent and the other to the hydroxypyridonering. The selection of a synthetic route for the preparation of aparticular compound will depend on various factors, including therelative availability of suitable intermediates.

It will be appreciated that the present invention extends to the variouscompounds, per se, obtainable by the specific examples of these threeapproaches described above for use in connection with the3-hydroxypyridones.

In the case of the 1-hydroxypyrid-2-ones it is not possible to effectlinkage of the rings through the nitrogen atoms thereof and linkage istherefore effected at one of the carbon atoms of the ring. The preferredapproach to such linking is analogous to the second approach describedabove for the linking of 3-hydroxypyrid-2- and -4-ones in that itinvolves the use of a 1-hydroxypyrid-2-one having a substituentcontaining a suitable functional group, for example at the 4-position ofthe ring, which is reacted with a reagent containing suitable functionalgroups for reaction therewith. The previous discussion in relation tothe second approach used with 3-hydroxypyrid-2- and -4-ones thereforeapplies also in the present case, with the modification that groups suchas --(CH₂)_(i) --CO₂ H or --(CH₂)_(i) --NH₂, wherein i is as definedhereinbefore, are more usually attached to a ring carbon atom of a1-hydroxypyrid-2-one through another group, particularly an --O-- group,in view of the greater ease of synthesising aminoalkoxy andcarboxyalkoxy substituted 1-hydroxypyrid-2-ones as compared with theaminoalkyl and carboxyalkyl substituted compounds. The synthesis of suchsubstituted 1-hydroxypyrid-2-ones is discussed in detail in theco-pending applications of even date referred to hereinbefore but it mayconveniently be achieved using a compound such as2-chloro-4-nitropyridine-1-oxide for example, which may be subjected tonucleophilic substitution to replace the nitro group by a substitutedalkoxy group which corresponds to or is convertible to that required,the chloro group then being converted to a hydroxy group by basichydrolysis. It will be appreciated that the present invention extends tothe various compounds, per se, obtainable using the specific reagentsreferred to above in connection with the second approach for the linkingof 3-hydroxypyrid-2-and -4-ones when used in conjuction with a1-hydroxypyrid-2-one which is C-substituted by a group --O--(CH₂)_(i)--CO₂ H or --O--(CH₂)_(i) --NH₂, wherein i is as defined hereinbeforeand is preferably greater than 1 in the case of the latter type ofgroup. The carboxy group will be in activated form in the former case,and both types of group may conveniently be substituted at the4-position of the 1-hydroxypyrid-2-one.

Whilst it will usually be most convenient to use the compounds accordingto the present invention in the normal form, they may of course be usedin salt form if desired, salts being formed between a physiologicallyacceptable cation and the anion formed by the loss of a proton from thering hydroxy group. Moreover, appropriate substituent groups may ofcourse be in salt form.

The synthesis of compounds according to the present invention and ofvarious reagents providing linking groups between the hydroxypyridonerings is illustrated hereinafter in the Examples. Compounds useful asreagents in the preparation of linking groups containing amide functionsmay readily be prepared from suitable amine precursors by reaction withthe appropriate acylating agent, conveniently in the form of anactivated ester. Thus, for example, the amineN-(2-aminoethyl)-1,3-propanediamine used in Example 6 may be reacted, asdescribed in the footnote (1)(A) and (B) to that Example, with glycinein activated ester form and containing a protected amino group, forexample as N-benzylglycine p-nitrophenyl ester, to provide, afterremoval of the N-protecting group, the compound H₂ N--CH₂ CONH--(CH₂)₂--NH--(CH₂)₃ --NHCOCH₂ --NH₂ or the compound H₂ N--CH₂ CONH--(CH₂)₂--N(COCH₂ --NH₂)--(CH₂)₃ --NHCOCO₂ --NH₂, depending on the conditionsused. Thus, in ethyl acetate and like solvents the former compound willprecipitate from the reaction mixture and the latter compound is notformed even in the presence of a three, rather than two, molarproportion of the glycine derivative. However, in a more polar solvent,such as dimethylformamide, a three molar proportion will lead to theformation of the latter compound. The former compound may be used assuch in the preparation of two ring compounds or, alternatively, may bereacted in a solvent such as diamethylformamide with another amino acid.Thus, reaction with a suitable β-alanine derivative, for example, willlead to the formation of the compound H₂ N--CH₂ CONH--(CH₂)₂ --N(COCH₂CH₂ --NH₂)--(CH₂)₃ --NHCOCH₂ --NH₂.

It will be appreciated that the procedures which have been described andillustrated herein with particular reference to the production of tworing and three ring compounds may be applied with appropriatemodification for the production of compounds containing more rings thanthis. Moreover, it will be appreciated that the routes described are notthe only routes available to the compounds of the present invention andthat various alternatives may be used as will be apparent to thoseskilled in the art.

The compounds may be formulated for use as pharmaceuticals forveterinary, for example in an avian or especially a mammalian context,or particularly human use by a variety of methods. For instance, theymay be applied as an aqueous, oily or emulsified compositionincorporating a liquid diluent which most usually will be employed forparenteral administration and therefore will be sterile and pyrogenfree. However, it will be appreciated from the foregoing discussion inrelation to desferrioxamine that oral administration is to be preferred,and the compounds of the present invention may be given by such a route.Although compositions incorporating a liquid diluent may be used fororal administration, it is preferred to use compositions incorporating asolid carrier, for example a conventional solid carrier material such asstarch, lactose, dextrin or magnesium stearate, the oral compositionthen conveniently being of a formed type, for example as tablets,capsules (including spansules), etc.

Other forms of administration than by injection or through the oralroute may also be considered in both human and veterinary contexts, forexample other forms known in the art such as the use of suppositories orpessaries, particularly for human administration.

Compositions may be formulated in unit dosage form, i.e. in the form ofdiscrete portions each comprising a unit dose, or a multiple orsub-multiple of a unit dose. Whilst the dosage of active compound givenwill depend on various factors, including the particular compound whichis employed in the composition, it may be stated by way of guidance thatsatisfactory control of the amount of iron present in the human bodywill often be achieved using a daily dosage of about 0.1 g to 5 g,particularly of about 0.5 g to 2 g, veterinary doses being on a similarg/kg body weight ratio. However, it will be appreciated that it may beappropriate under certain circumstances to give daily dosages eitherbelow or above these levels. Where desired, more than one compoundaccording to the present invention may be administered in thepharmaceutical composition or, indeed, other active compounds may beincluded in the composition.

The 3-hydroxypyrid-2-ones, 3-hydroxypyrid-4-ones and1-hydroxypyrid-2-ones all possess a high affinity for iron(III), asevidenced by log K_(sol) values (log K_(sol) is defined as being equalto log β_(Fe)(L)n +21-[pK_(sp) +n log a_(L) (H⁺)+m log a_(L) (Ca⁺⁺)]where log β_(Fe)(L)n is the cumulative affinity constant of the ligandin question for iron(III), pK_(sp) is the negative logarithm of thesolubility product for Fe(OH)₃ and has a value of 39, n and m are thenumber of hydrogen and calcium ions, respectively, which are bound tothe ligand, and a_(L) (H⁺) and a_(L) (Ca⁺⁺) are the affinities of theligand for hydrogen ions and calcium ions, respectively). In order tosolubilise iron(III) hydroxide, log K_(sol) must be greater than 0 andin order to remove iron from transferrin, log K_(sol) should be inexcess of 6.0. The log K_(sol) values for the single ring compounds3-hydroxyl-1-methylpyrid-2-one and 1,2-dimethyl-3-hydroxypyrid-4-one, byway of example, are 10.0 and 9.5, respectively, thus comparingfavourably with those of the bidentate hydroxamates at about 4.0, ofcatechols at about 8.0, of desferrioxamine at 6.0, and ofdiethylenetriamine penta-acetic acid (DTPA) at 2.0.

The compounds of the present invention have certain advantages, ascompared with the single ring compounds of the earlier UK patentapplications referred to hereinbefore, in binding with metals. Inparticular, they tend to have higher stability constants therebyremoving the need to use additional substituent groups to increasestability. This enhanced stability is due to the chelate effect, so thatthe reaction of, for example, a three ring hexadentate compound withiron(III) will involve a greater increase in entropy than with theequivalent single ring bidentate compound, whereas the enthalpy increasewill be similar in each case. The superiority of the hexadentate ligandis clearly demonstrated by the "dilution effect", the fraction ofiron(III) bound to the hexadentate compound being relatively independentof dilution, whereas with the bidentate compound, the binding isstrongly dependent on dilution. The hexadentate compounds of the presentinvention will thus generally be superior to the corresponding singlering bidentate compound for combination with free iron. However, thesize of the hexadentate compounds in some cases may interfere with theirability to combine with bound iron in ferritin, and consequently some ofthe single ring bidentate compounds may be more effective at removingiron from ferritin than the corresponding hexadentate ligand. Thecompounds are however fully effective at removing iron from transferrinand as such removal is effected a transfer will occur in the body ofiron bound as ferritin to iron bound and transferrin, thus allowing theremoval of that iron by the compounds. Although the lower efficiency ofthe compounds of the present invention, as compared with such singlering compounds, at removing iron from ferritin therefore presents noreal problem, it may be advantageous in some cases to use the compoundsof the present invention in admixture with a single ring compound, whichmay contain the same form of ring as in the linked compound with thesame type of carbon-substitution or lack thereof, the same form of ringdifferently substituted or a different type of ring system, suitablesingle ring compounds being those described in the aforementioned threeearlier UK patent applications and their equivalents. Particularly ofinterest are mixtures of compounds according to the present inventioncontaining three 3-hydroxypyrid-2- or -4-one rings and a single ringcompound of the same type substituted on the nitrogen atom thereof andoptionally on one or more ring carbon atoms by an aliphatic hydrocarbongroup of 1 to 6 carbon atoms, such single ring compounds being thosedescribed in UK Patent Application GB 2118176A (U.S. application Ser.No. 933,338, U.S. Pat. No. 4,840,958) and its equivalents. Such mixturesof three ring and single ring compounds will exhibit very efficient ironremoval characteristics, probably on the basis of free iron being takenup mainly by the three ring compounds and bound iron mainly by thesingle ring compounds from which it may then be abstracted by the threering compounds.

In addition to the use described hereinbefore for the treatment ofgeneral iron overload, the linked hydroxypyridone compounds describedherein are also for interest for use in certain pathological conditionswhere there may be an excess of iron deposited at certain sites eventhough the patient does not exhibit a general iron overlaod, this beingthe case, for example, in certain arthritic and cancerous conditions.Indeed in some patients having such conditions, the patient may exhibitan overall anaemia and the metal-free linked hydroxypyridone compoundsmay then be used in conjunction with an iron complex which will correctthe overall anaemia whilst the metal-free compound will act to removeiron from pathological to physiological sites. This iron complex may beof various types but of particular interest are the iron complexes, andespecially the 3:1 neutral iron(III) complexes, of the single ringcompounds described in the aforementioned three earlier UK patentapplications and their equivalents. These compounds may contain the sameform of ring as in the linked compound with the same type of carbonsubstitution or lack thereof, the same form of ring differentlysubstituted, or a different type of ring system. The aliphatichydrocarbon group-substituted 3-hydroxypyrid-2- and 4-one compoundsreferred to above are again of particular interest. Alternatively, themetal-free linked hydroxypyridones may be used in conjunction with andiron complex which is a complex of such a linked hydroxypyridone, whichmay be the same or different, such linked hydroxypyridone iron complexesand their use in this context being discussed in detail hereinafter.

Although the major use of the metal-free compounds of the presentinvention is in the removal of iron, they are also of potential interestfor the removal of some other metals which may be present in the body indeleterious amounts. Indeed, the compounds of the present invention areperhaps of greater interest in this respect than the single ringcompounds as the ability to incorporate differing numbers of rings intothe compounds allows them to be tailored to be particularly applicablefor use with different metals. Thus the three ring hexadentate compoundsare particularly suited to the removal of trivalent cations such asiron(III) or aluminium(III) with which they form a neutral 1:1 complex.On the other hand, the two ring tetradentate compounds are particularlysuited to the removal of divalent cations such as copper(II) andmagnesium(II), and the four ring octadentate compounds are particularlysuited to the removal of tetravalent cations such as plutonium(IV) andother related transuranic metals. Alternatively, compounds containingmultiples of these numbers of rings may be of interest for binding withmore than one metal atom so that, for example, a six ring compound maybe used to form a neutral complex by binding with two iron atoms in theferric form.

The present invention thus includes the use of a compound as definedhereinbefore for use in medicine, for example for the removal from thebody of toxic amounts of metals, including copper, aluminium andparticularly iron. Moreover, the invention also includes a method forthe treatment of a patient having toxic amounts of a metal, for examplecopper, aluminium and particularly iron, in the body which comprisesadministering to said patient an amount of compound as definedhereinbefore to effect a reduction of the levels of this metal in thepatient's body.

Uses of the compounds of the present invention for combination withmetals other than iron may extend to the treatment of body fluidsoutside the body or even to quite other contexts than the treatment ofpatients. One particular area of some interest involves the treatment ofpatients on haemodialysis who may show a dangerous build up of aluminiumin the body. For the treatment of such patients the compounds of thepresent invention may conveniently be insolubilised by attachment to asupport material and then contacted with the patient's blood to removealuminium therefrom. Other uses of the compounds extending beyond theremoval of metals from the body and involving particularly plutonium,for example its recovery from waste materials, or even iron, maysimilarly conveniently require insolubilisation of the compound byattachment to a support material.

The support material used may conveniently be one of various types ofpolymer described in the art for use in similar contexts, for example acarbohydrate material which may be of an agarose, dextran or other type,or a polystyrene or other material such as is used in ion-exchangeresins. Various approaches known in the art may be used for effectingattachment of the compounds to such support materials but it ispreferred to effect such attachment through the linking groups althoughit is possible to use another substituent group on the hydroxypyridonering such as an acidic or basic group which forms an amide type oflinkage through reaction with a complementary group on the supportmaterial. Convenient approaches involving attachment through the linkinggroups are direct conjugation using substituent carbodiimides orindirect methods using labile p-nitrophenol or N-hydroxysuccinimideesters. However a wide range of other procesures is generally available,for example using acid anhydrides and a variety of bifunctionalreagents.

Just as iron overload can pose problems in some patients, irondeficiency anaemia can pose problems in others. As well as being ofvalue as the metal-free compounds for the treatment of conditionsinvolving iron overload, the linked hydroxypyridones describedhereinbefore are of interest in the iron complex form for the treatmentof iron deficiency anaemia.

An adequate supply of iron to the body is an essential requirement fortissue growth in both man and animals. Although there is normally anample amount of iron in the diet, the level of absorption of iron fromfood is generally low so that the supply of iron to the body can easilybecome critical under a variety of conditions. Iron deficiency anaemiais commonly encountered in pregnancy and may also present a problem inthe newly born, particularly in certain animal species such as the pig.Moreover, in certain pathological conditions there is a mal distributionof body iron leading to a state of chronic anaemia. This is seen inchronic diseases such as rheumatoid arthritis, certain haemolyticdiseases and cancer.

Although a wide range of iron compounds is already marketed for thetreatment of iron deficiency anaemia, the level of iron uptake by thebody from these compounds is often quite low, necessitating theadministration of relatively high dosage levels of the compound. Theadministration of high dose, poorly absorbed, iron complexes may causesiderosis of the gut wall and a variety of side effects such as nausea,vomiting, constipation and heavy malodorous stools. We have now foundthat the iron complexes of the linked hydroxypyridones describedhereinbefore are of particular value in the treatment of suchconditions.

Accordingly the present invention further comprises an iron complex of acompound in which two or more rings, being a 3-hydroxypyrid-2-one,3-hydroxypyrid-4-one or 1-hydroxypyrid-2-one, are linked.

The comments made hereinbefore in relation to K_(part) values for themetal-free compounds and their corresponding iron complexes in the caseof preferred compounds apply equally to the selection of preferredmetal-free compounds and of preferred iron complexes. The comments madehereinbefore with regard to preferences as to the nature and position oflinking groups and other substituents thus apply equally in relation tothe iron complexes.

The iron complexes present in the pharmaceutical compositions accordingto the present invention preferably contain iron in the ferric state.Although the use of complexes containing iron in the ferrous state maybe considered, such complexes tend to be less stable and are thus ofless interest. The iron complexes are preferably neutral, i.e. therebeing an internal balance of charges between the metal cation or cationsand the ligand or ligands bound covalently thereto without the necessityfor the presence of a non-covalently bound ion or ions, for example achloride ion, to achieve balance. Moreover, the use of linkedhydroxpyridones containing ionisable substituent groups (or linkinggroups) is of less interest and it is preferred that this internalbalance of charges is achieved by complexing with an iron cation orcations a compound containing the appropriate number of hydroxypyridonerings to thereby provide, by the loss of a hydroxy proton from eachring, the number of anionic groups O⁻ necessary to neutralise the chargeon the cation or cations. Although such neutrality may be achieved, forexample, in a complex containing a 3:2 molar proportion of an anion froma two ring compound:iron(III), preferred neutral complexes of this typeare those formed between the anion derived from a compound containingthree, or a multiple of three, hydroxypyridone rings and a ferriccation, or the same multiple of ferric cations, particularly thosecontaining a trivalent anion derived from a three ring compound and asingle ferric cation. It will be appreciated, however, that theinvention does not exclude the use of complexes in which the charge onthe anion derived from the linked hydroxypyridone compound and that onthe ferric cation or cations do not fully neutralise each other, forexample owing to the presence of an ionisable linking group, so thatassociation with a further non-covalently bound physiologicallyacceptable ion or ions is necessary to achieve a balance of charges,although such complexes are generally of lesser interest.

The present invention thus particularly includes a neutral iron complexcontaining 1 molar proportion of iron(III) and 1 molar proportion of acompound in which three rings, being a 3-hydroxypyrid-2-one,3-hydroxypyrid-4-one or 1-hydroxypyrid-2-one, are linked. The use ofneutral iron complexes according to the present invention is discussedhereinafter with particular reference to such 1:1 complexes.

The iron complexes are conveniently prepared by the reaction of thelinked hydroxypyridone compound and iron ions, the latter convenientlybeing derived from an iron salt, particularly a ferric halide andespecially ferric chloride. The reaction is conveniently effected in asuitable mutual solvent and water may often be used for this purpose. Ifdesired, however, an aqueous/organic solvent mixture may be used or anorganic solvent, for example ethanol, methanol, chloroform and mixturesof these solvents together and/or with water where appropriate. Inparticular, methanol or especially ethanol may be used as the solventwhere it is desired to effect the separation of at least a major part ofa by-product such as sodium chloride by precipitation whilst the ironcomplex is retained in solution. Alternative procedures may, however, beused and will be apparent to those skilled in the art.

It will be appreciated that the nature of the iron complex obtained bythe reaction of a linked hydroxypyridone compound and iron ions willdepend both on the proportion of these two reactants and upon the pH ofthe reaction medium. Thus, for the preparation of a 1:1 ferric complex,for example, the linked three ring hydroxypyridone compound and theferric salt are conveniently mixed in solution in a 1:1 molar proportionand the pH adjusted to a value in the range of 6 to 9, for example 7 or8. Adjustment of the pH may conveniently be effected by the additioneither of sodium carbonate or of a hydroxide base such as sodium orammonium hydroxide, the use of a hydroxide base being of particularinterest when preparing the iron complexes in batches of 20 g or more.When using a hydroxide base, the reaction may conveniently be carriedout in a medium containing water as the solvent, for example in water oran ethanol:water mixture, and the pH adjusted by the addition of a 2molar aqueous solution of the base. It will be appreciated that thepresence of water in the reaction mixture will lead to the retention ofa by-product in the iron complex on evaporation of the solvent (achloride where the iron salt is ferric chloride). However, this can beremoved, if desired, by procedures such as crystallisation from asuitable solvent system or sublimation in the particular case ofammonium chloride.

Reaction to form the iron complex is generally rapid and will usuallyhave proceeded substantially to completion after 5 minutes at about 20°C., although a longer reaction time may be used if necessary. Followingseparation of any precipitated by-product, such as sodium chloride inthe case of certain solvent systems, the reaction mixture mayconveniently be evaporated on a rotary evaporator or freeze dried toyield the solid iron complex. This may, if desired, be crystallised froma suitable solvent, for example water, an alcohol such as ethanol, or asolvent mixture, including mixtures containing an ether. The presentinvention thus further includes a process for the preparation of an ironcomplex of a linked hydroxypyridone compound as defined hereinbeforewhich comprises reacting said compound with iron ions and isolating theresultant complex.

Whilst for some uses it may be appropriate to prepare the iron complexin substantially pure form, i.e. substantially free from by-products ofmanufacture, in other cases, for example with a solid oral formulationas described hereinafter, the presence of by-products such as sodiumchloride may be quite acceptable. In general, however, the neutral 1:1[linked hydroxypyridone compound:iron(III)] complex is of particularinterest in a form free from by-products which are complexes containingdifferent proportions of hydroxypyridone and iron. As indicatedhereinafter, it may be advantageous under some circumstances for theiron complex to be used in admixture with the metal-free linkedhydroxypyridone compound and, if desired, such a mixture may be obtaineddirectly by reacting a molar proportion of the compound and iron ions ofgreater than 1:1.

The iron complexes may be formulated as pharmaceuticals for veterinary,for example in an avian or particularly a mammalian context, or humanuse by a variety of methods and the invention includes a pharmaceuticalcomposition comprising an iron complex as hereinbefore defined togetherwith a physiologically acceptable diluent or carrier. The comments madehereinbefore with regard to the formulation of the metal-free compoundsapply equally to the iron complexes, although in this instancecompositions for parenteral administration are of greater interestparticularly in the context of animal treatment. The problems of irondeficiency anaemia in newly born pigs arise primarily during the firstthree weeks or so of their life when a very rapid weight gain takesplace. The iron complexes of the present invention may be used to treatpiglets directly by a parenteral route, for example intramuscular, ororal, for example as a liquid preparation "injected into the mouth".However, an alternative approach is to enhance the iron content of themilk on which the piglets are feeding by treating the mother pig usingoral or parenteral administration, for example an injectable slowrelease preparation (such an approach may also be an interest in a humancontext). When it is applicable to feed piglets on foodstuffs other thanthe milk of the mother pig, it may also be possible to effect thepharmaceutical administration of the iron complex in this otherfoodstuff.

As with the metal-free compounds, the dosage of the hydroxypyridone ironcomplex which is given will depend on various factors, including theparticular compound which is employed in the composition. It may bestated by way of guidance, however, that maintenance of the amount ofiron present in the human body at a satisfactory level will often beachieved using a daily dosage, in terms of the iron content of thecompound, which lies in a range from about 0.1 to 100 mg and often in arange from 0.5 to 10 mg, for example 1 or 2 mg, veterinary doses beingon a similar g/Kg body weight ratio. However, it will be appreciatedthat it may be appropriate under certain circumstances to give dailydosages either below or above these levels. In general, the aim shouldbe to provide the amount of iron required by the patient withoutadministering any undue excess and the properties of the pharmaceuticalcompositions according to the present invention are particularly suitedto the achievement of this aim. Where desired, an iron complex of morethan one linked hydroxypyridone compound as described above may bepresent in the pharmaceutical composition or indeed other activecompounds may be included in the composition, for example compoundshaving the ability to facilitate the treatment of anaemia, such as folicacid. Another additional component which may be included in thecomposition, if desired, is a source of zinc. Iron compounds used in thetreatment of iron deficiency anaemia can inhibit the mechanism of zincuptake in the body and this can cause serious side effects in the foetuswhen treating anaemia in a pregnant female. It is believed, however,that the iron complexes of the present invention have a furtheradvantage in that they either do not have this effect or exhibit theeffect at a lower level than the compounds at present used in thetreatment of anaemia. Accordingly, it may often be the case that thelevel of zinc providing compound added to the composition may notrequire to be high or, with preferred formulations of the ironcomplexes, may be dispensed with altogether.

It has never before been appreciated that iron complexes such as thosedescribed herein might be used in a pharmaceutical context. Accordinglythe present invention includes an iron complex defined hereinbefore foruse in medicine, particularly in the treatment of iron deficiencyanaemia (in the broad sense of this term).

We have found that the iron complexes described herein are of value inthe treatment of iron deficiency anaemia both in humans and also in aveterinary context, particularly for the treatment of various mammalianspecies and especially pigs. The complexes will partition into n-octanolindicating that they are able to permeate biological membranes, thisproperty being confirmed in practice by tests of the ability of the ⁵⁹Fe labelled iron complexes to permeate erythrocytes. The ability of thecompounds in this respect will depend on the nature of thesubstituent(s) present therein and the reflection of this ability in theK_(part) values of various compounds has been referred to hereinbefore.Once present in the bloodstream, the complexes will donate iron totransferrin, a position of equilibrium being set up between thecomplexes and transferrin. It is because of the existence of thisequilibrium that the corresponding metal-free linked hydroxypyridonecompound may equally be used in the treatment of iron overload, althoughcertain of these compounds may be of particular value for use in thefree state for iron removal and others may be of particular value foruse as iron complexes for iron supply.

Certain aspects of their formulation may enhance the activity of thecomplexes in particular contexts. Thus, the neutral 1:1 ferric complexesare of particular value as being stable over a wide pH range from about4 or 5 up to 10 and even at the pH values of less than 4 prevailing inthe stomach free iron should not be liberated. Thus, it has beenobserved that, even at pH 0.5, the iron of such a 1:1 complex is stillbound although even though not necessarily by all six of the originalcovalent bonds. Moreover, when the complex is cleared from the stomachand reaches the small intestine any internal dissociation of the bondswithin the complex should be reversed under the alkaline conditionsprevailing therein. Protection of the iron complexes from the acidicconditions of the stomach should not therefore be necessary from thepoint of view of preventing the liberation of iron from the complex. Itis a possible with some forms of linking group, however, that breakdownof the group may occur under the acidic conditions prevailing in thestomach, for example by cleavage of an amide group. In suchcircumstances, the use of a method of formulation which avoids orreduces exposure of the iron complex to the acidic conditions of thestomach can be of value. Such an approach may involve various types ofcontrolled release system, ranging from one, which may for example bebased on a polymer, which simply provides a delayed release of thecomplex with time, through a system which is resistant to dissociationunder acidic conditions, for example by the use of buffering, to asystem which and is biased towards release under conditions such asprevail in the small intestine, for example a pH sensitive system whichis stabilised towards a pH of 1 to 3 such as prevails in the stomach butnot one of 7 to 9 such as prevails in the small intestine. Since the pHof the stomach is higher after a meal, it may be advantageous, whatevermethod of formulation is used, to administer the iron complexes at sucha time.

A particularly convenient approach to a controlled release compositioninvolves encapsulating the iron complex by a material which is resistantto dissociation in the stomach but which is adapted towards dissociationin the small intestine (or possibly, if the dissociation is slow, in thelarge intestine). Such encapsulation may be achieved with liposomes,phospholipids generally being resistant to dissociation under acidicconditions. The liposomally entrapped 1:1 iron(III) complexes cantherefore survive the acid environment of the stomach withoutdissociation. On entry into the small intestine, the pancreatic enzymesrapidly destroy the phospholipid-dependent structure of the liposomesthereby releasing the 1:1 complex. Liposome disruption is furtherfacilitated by the presence of bile salts. However, it is usually moreconvenient to effect the encapsulation, including microencapsulation, bythe use of a solid composition of a pH sensitive nature.

The preparation of solid compositions adapted to resist dissociationunder acidic conditions but adapted towards dissociation undernon-acidic conditions is well known in the art and most often involvesthe use of enteric coating, whereby tablets, capsules, etc, or theinidividual particles or granules contained therein, are coated with asuitable material. Such procedures are described, for example, in thearticle entitled "Production of enteric coated capsules" by Jones inManufacturing Chemist and Aerosol News, May 1970, and in such standardreference books as "Pharmaceutical Dosage Forms, Volume III byLiebermann and Lackmann (published by Marcel Decker). One particularmethod of encapsulation involves the use of gelatine capsules coatedwith a cellulose acetate phthalate/diethylphthalate layer. This coatingprotects the gelatin capsule from the action of water under the acidconditions of the stomach where the coating is protonated and thereforestable. The coating is however destabilised under the neutral/alkalineconditions of the intestine where it is not protonated, thereby allowingwater to act on the gelatin. Once released in the intestine the rate ofpermeation of the intestine wall by the water soluble 1:1 iron(III)complex is relatively constant irrespective of the position within theintestine, i.e. whether in the jejunum, ileum or large intestine. Otherexamples of methods of formulation which may be used include the use ofpolymeric hydrogel formulations which do not actually encapsulate theiron complex but which are resistant to dissociation under acidicconditions.

Another aspect of the formulation of the iron complexes to confercertain particular advantages is the use of a metal-free linkedhydroxypyridone compound in admixture with its iron complex. Thus, asmentioned hereinbefore, in certain pathological conditions there may bean excess of iron deposited at certain sites even though the patientexhibits an overall anaemia. In patients having such conditions the useof such a mixture has the advantage that the iron complex will remedythe overall anaemia whilst the free linked hydroxypyridone compound willact to remove iron from pathological to physiological sites. Moreover,there may be an advantage in formulating the iron complex of onecompound as described herein with another one of such compounds in themetal-free form. Thus, it is preferable for the linked hydroxypyridonecompound present in an iron donor to be rapidly metabolized so as toeffect its removal from the system once it has given up its iron at anappropriate site in the system, whilst it is preferable for a linkedhydroxypyridone compound being used as an iron remover not to be rapidlymetabolized so that it remains in the system, taking up iron, for anextended period. For this reason the use of different linkedhydroxypyridone compounds in the free form and as the iron complex hascertain advantages. Moreover, different compounds may, for otherreasons, function more efficiently either in the free form as an ironremover or in complex form as an iron donor. If desired, the freecompound may alternatively be used in the form of a salt formed with theanion produced by the loss of a hydroxy proton and containing aphysiologically acceptable cation, for example as describedhereinbefore. As an alternative to combination with a differentmetal-free linked hydroxypyridone compound of the same type, the ironcomplex may be used in combination with another iron chelating agent.

When using such a mixture of an iron complex of a linked hydroxypyridonecompound and a metal-free compound, the daily dosage of the iron complexmay be as previously described and the daily dosage of the free compoundmay also be that described in relation to the use of such compounds iniron overload conditions.

It will be appreciated that the present invention also includes a methodfor the treatment of a patient which comprises administering to saidpatient an amount of an iron complex as described hereinbefore in orderto effect an increase in the levels of iron in the patient's bloodstream.

In addition to the pharmaceutical uses of the iron complexes discussedabove they are also of potential interest as a source of iron in variousother contexts including in cell and bacterial growth, in plant growth,as a colouring agent and in the control of iron transport acrossmembranes.

The invention is illustrated by the following Examples which describethe preparation of various compounds of the form ##STR13## wherein R, R"and R" each represent a substituted or unsubstituted3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one or 1-hydroxypyrid-2-one ring,n represents 0 or an integer 1, 2, 3, 4 etc. (no rings R" being presentwhen n is 0) and L represents a linking group.

The exemplified compounds contain two or three 3-hydroxypyrid-2-one or3-hydroxy-2-methylpyrid-4-one rings joined by a variety of linkinggroups. For greater ease of reference the different compounds describedin the Examples are illustrated in Table 1, the numbering of thecompounds corresponding to that of the Examples. It will be appreciatedthat further compounds may be produced by using in the procedures ofExamples 2, 3, 7 and 9 the corresponding 3-hydroxypyrid-2-one reagentwith the same linking group reagents as described in these Examples andin the procedures of Examples 4, 5, 8 and 10 the corresponding3-hydroxy-2-methylpyrid-4-one reagent with the same linking reagents asdescribed in these Examples. Such further specific compounds comprising3-hydroxypyrid-2-one rings with a linking group such as is present incompounds 2, 3, 7 and 9 and 3-hydroxy-2-methylpyrid-4-one rings with alinking group such as is present in compounds 4, 5, 8 and 10 are thusalso individually included within the scope of the present invention.

It will also be noted that, as discussed hereinbefore in relation to thepreparation of amine reactants, a change in the nature of the solventused can produce a different reaction product. Thus, in Example 7 wheremethylene chloride is used, the secondary amine group in the reactantdoes not react with the active ester, while in Example 8 where the morepoler dimethylformamide is used as the solvent, the secondary aminegroup in the reactant does react with the active ester. It will beappreciated therefore that different products containing three ratherthan two rings, or vice versa, may be obtained in the procedures ofExamples 7, 8, 9 and 10 by using different reaction conditions and thatthese alternative compounds are also individually included within thescope of the present invention.

                                      TABLE 1                                     __________________________________________________________________________          Number                                                                  Compound                                                                            of rings                                                                           Ring system                                                                            Linking group                                             __________________________________________________________________________    1     2                                                                                   ##STR14##                                                                             CH.sub.2(CH.sub.2).sub.6CH.sub.2                          1A    2                                                                                   ##STR15##                                                                             CH.sub.2(CH.sub.2).sub.7CH.sub.2                          2     2                                                                                   ##STR16##                                                                             (CH.sub.2).sub.2CONH(CH.sub.2).sub.8NHCO(CH.sub.2).sub                        .2                                                        3     2                                                                                   ##STR17##                                                                             (CH.sub.2).sub.5CONH(CH.sub.2).sub.2NHCO(CH.sub.2).sub                        .5                                                        3A    2                                                                                   ##STR18##                                                                             (CH.sub.2).sub.3 CONH(CH.sub.2).sub.2NHCO(CH.sub.2).su                        b.3                                                       3B    2                                                                                   ##STR19##                                                                             CH.sub.2 CONH(CH.sub.2).sub.2NHCOCH.sub.2                 4     2                                                                                   ##STR20##                                                                             CH.sub.2 CONH(CH.sub.2).sub.6NHCOCH.sub.2                 5     2                                                                                   ##STR21##                                                                             CH.sub.2 CONH(CH.sub.2).sub.8NHCOCH.sub.2                 6     2                                                                                   ##STR22##                                                                             (CH.sub.2).sub.3NH(CH.sub.2).sub.2                        6A    2                                                                                   ##STR23##                                                                             CH.sub.2 CONH(CH.sub. 2).sub.2NH(CH.sub.2).sub.3NHCOCH                        .sub.2                                                    6B    3                                                                                   ##STR24##                                                                              ##STR25##                                                6C    3                                                                                   ##STR26##                                                                              ##STR27##                                                7     2                                                                                   ##STR28##                                                                             (CH.sub.2).sub.2 CONH(CH.sub.2).sub.4NH(CH.sub.2).sub.                        3NHCO(CH.sub.2).sub.2                                     8     3                                                                                   ##STR29##                                                                              ##STR30##                                                9     3                                                                                   ##STR31##                                                                              ##STR32##                                                10    3                                                                                   ##STR33##                                                                              ##STR34##                                                __________________________________________________________________________

EXAMPLES EXAMPLE 1 Preparation of1,8-di-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)-octane and other relatedcompounds

3-Hydroxy-2-methyl-4-pyrone (22.2 g) in methanol (225 ml) is added toaqueous sodium hydroxide (25 ml H₂ O containing 7.5 g NaOH). Benzylchloride (25.5 g) is added and the mixture is refluxed for 6 hours andis then allowed to cool overnight. The bulk of the methanol is removedunder vacuum and the residue is treated with water (50 ml). The mixtureis extracted into dichloromethane (3×25 ml). The extracts are combined,washed with 5% w/v NaOH (2×25 ml), then water (2×25 ml) and dried overmagnesium sulphate. Evaporation of the solvent gives crude3-benzyloxy-2-methyl-4-pyrone or "benzyl maltol" (35 g, 92%) which ispurified by distillation in nitrogen under reduced pressure to yield acolourless oil (28 g) of b.p. 148° C./0.2 mm.

3-Benzyloxy-2-methyl-4-pyrone (0.066 moles) and 1,8-diaminooctane (0.022moles) are mixed in 2:1 v/v aqueous methanol (300 ml), solid sodiumhydroxide (2 g) is added, and the mixture is heated on a steam bath for5 hours. The mixture is allowed to cool and an aliquot is taken,acidified to pH 2 with concentrated hydrochloric acid and the solventremoved on the rotary evaporator to give a solid residue. This residueis checked for completion of the reaction through its n.m.r. spectrum,the presence of protons exchangeable at ca δ=8.3 (d₆ DMSO) showing thatthe reaction is not complete. If this is the case, refluxing iscontinued until the solid obtained from an aliquot shows no suchprotons. On complete reaction being found to have occurred, the mixtureis cooled and acidified to pH 2 with concentrated hydrochloric acid, anyprecipitate formed at this stage being dissolved by the addition ofmethanol to the mixture. Palladium/carbon catalyst is then added and thesolution is hydrogenated at room temperature and atmospheric pressure inorder to effect debenzylation. The catalyst is removed by filtration andthe solvent by rotary evaporation, the resulting solid then beingrecrystallised from water to give the title compound.sup.(1) in 40%yield as a white solid of m.p. 297°-300° dec; γ_(max) (nujol) 1340 and1620 cm⁻¹ ; δ(d₆ DMSO/D₂ O), 1.5 (m, 12H), 2.5 (s, 6H), 4.3 (m, 4H), 7.1(d, 2H) and 8.1 (d, 2H); m/e 360.

EXAMPLE 2 Preparation of1,8-di-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)propionamido]-octane

β-Alanine (5 g) in water (50 ml) is added to3-benzyloxy-2-methyl-4-pyrone (10 g) in 95% ethanol (100 ml) and sodiumhydroxide (4 g) in water (50 ml) is then slowly added to the reactionmixture until a pH of 13 is obtained. The solution is refluxed for 15minutes during which time the colour changes from yellow to amber. Aftercooling, the solution is acidified to pH 2.5 with concentrated HCl(about 2 ml) and is then rotary evaporated at 50° C. The resulting oilis subjected to a methylene chloride/water extraction. The solid whichremains is triturated with acetone and recrystallised from water to give3-benzyloxy-1-(2'-carboxyethyl)-2-methylpyrid-4-one, m.p. 81°-83° C.

The 3-benzyloxy-1-(2'-carboxyethyl)-2-methylpyrid-4-one (1 g) andN-hydroxysuccinamide (0.45 g) are dissolved in methylene chloride (7 ml)and dicyclohexyl carbodiimide (0.8 g) in methylene chloride (3 ml) isadded slowly at 0° C. with stirring. After 15 minutes the precipitatewhich has formed is separated by filtration and the filtrate is added toa solution of 1,8-diaminooctane (0.2 g) in methylene chloride (5 ml).The reaction mixture is then rotary evaporated to yield an oil which isdissolved in ethanol (30 ml) and hydrogenated over a palladium/charcoalcatalyst. Evaporation of the solution remaining after separation of thecatalyst yields an oil which is dissolved in acetone, the solution thenbeing treated with HCl gas. The resultant dihydrochloride salt which isprecipitated from solution is triturated with diethyl ether to give alow melting solid. This solid is further purified by chromatography onSephadex G10 to yield the title compound as the dihydrochloride salt inapproximately 50% yield, δ(d₆ DMSO) 1.1 (m, 8H), 1.9 (m, 4H), 2.5 (s,6H), 2.7 (m, 4H), 2.9 (m, 4H), 4.5 (t, 4H), 7.3 (d, 2H), 8.1 (d, 2H).

EXAMPLE 3 Preparation of1,2-di-[6-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)-hexanamido]-ethane andother related compounds

6-Aminocaproic acid is reacted with 3-benzyloxy-2-methyl-4-pyrone in anexactly analogous procedure to that described in Example 2 for thereaction of the latter compound with β-alanine to give3-benzyloxy-1-(5'-carboxypentyl)-2-methylpyrid-4-one in 70% yield, thiscompound being obtained after recrystallisation from ethanol/acetone(1:1 v/v) as white crystals, m.p. 145°-147° C.

3-Benzyloxy-1-(5'-carboxypentyl)-2-methylpyrid-4-one (1 g) andN-hydroxysuccinimide (0.4 g) are dissolved in methylene chloride (20 ml)and dicyclohexylcarbodiimide (0.7 g) in methylene chloride (5 ml) isadded slowly at 0° C. with stirring. After 20 minutes the resultingprecipitate is separated by filtration and the filtrate is treated withstirring with a solution of diaminoethane (0.092 g) in methylenechloride (1 ml). A flocculant precipitate is formed which is filtered togive a very hygroscopic solid which rapidly forms an oil. This oil isdissolved in 95% ethanol and hydrogenated over a platinum/charcoalcatalyst. The solution remaining after separation of the catalyst isrotary evaporated to yield an oil which is triturated with acetone andthen with diethyl ether to yield the title compound as a hygroscopicbuff solid, δ(d₆ DMSO) 1.0-1.7 (m, 12H), 2.0 (s, 6H), 2.2 (m, 4H), 3.0(m, 4H), 3.8 (m, 4H), 6.0 (d, 2H), 7.4 (d, 2H), 7.7 (m, 2H).

(1) In a variation of this procedure the compounds3-benzyloxy-1-(3'-carboxypropyl)-2-methylpyrid-4-one and3-benzyloxy-1-carboxymethyl-2-methylpyrid-4-one are prepared in ananalogous manner using 4-aminobutyric acid and glycine, respectively,then converted to the active ester and reacted with diaminoethane togive (A)1,2-di-[4-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)-butanamido]-ethane and(B) 1,2-di-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-ethane,respectively.

EXAMPLE 4 Preparation of1,6-di-(3-hydroxy-2-oxopyrid-1-ylacetamido)-hexane

2,3-Dihydroxypyridine (10 g) is suspended in ethylbromoacetate (40 ml)and the mixture is heated in a sealed tube for 24 hours at 140° C. Thetube is then cooled in solid CO₂ and opened. The contents are subjectedto rotary evaporation at 50° C. to yield a yellow solid.Recrystallisation of this solid from water yields1-ethoxycarbonylmethyl-3-hydroxypyrid-2-one as white crystals (10.8 g),m.p. 141°-151° C.

1-Ethoxycarbonylmethyl-3-hydroxypyrid-2-one (10 g) is dissolved inmethanol/water (9:1 v/v) (400 ml). To this solution is added benzylchloride (3 molar excess) and NaOH until the pH is above 12. The mixtureis then refluxed for six hours to give a clear orange solution. Themethanol is removed by rotary evaporation and the aqueous solution isextracted with dichloromethane to remove excess benzyl chloride. Theaqueous phase is diluted slightly by adding extra water and thenacidified to pH 2 using concentrated hydrochloric acid which results inthe precipitation of a beige solid. The mixture is cooled and theprecipitate filtered off and washed with diethyl ether. The crudeproduct is recrystallised from ethanol to give3-benzyloxy-1-carboxymethylpyrid-2-one (5.4 g, 41%), m.p. 176°-177° C.

3-Benzyloxy-1-carboxymethylpyrid-2-one (0.5 g) and N-hydroxy-succinimide(0.25 g) are dissolved in dimethylformamide (10 ml) and the solutioncooled in an ice bath. Dicyclohexylcarbodiimide (0.45 g) dissolved indimethylformamide (10 ml), is added to the pyridone and allowed to standfor 18 hours. The precipitated dicyclohexylurea is separated byfiltration and the filtrate evaporated to dryness. The resultant residueis recrystallised from a methylene chloride/diethylether mixture toyield 3-benzyloxy-1-succinimyloxycarbonylmethylpyrid-2-one (0.56 g,80%), m.p. 183°-184° C.

3-Benzyloxy-1-succinimyloxycarbonylmethylpyrid-2-one (0.5 g) isdissolved in dimethylformamide (12.5 ml). To this solution is added asolution of 1,6-diaminohexane (0.5 molar equivalent) in methanol (5 ml).The resulting white precipitate is separated by filtration and thecolourless filtrate is evaporated to dryness. The resultant residue isextracted with a chloroform/water mixture, the organic layer being dried(Na₂ SO₄) and evaporated to dryness. The resultant residue is dissolvedin ethanol containing 1% v/v of acetic acid and hydrogenated overpalladium charcoal. Filtration, evaporation and recrystallisation fromethanol yields the title compound as a white crystalline solid in 70%yield, m.p. 240°-242° C.; ν_(max) (nujol) 1645, 1580, 1555 cm¹ ; δ(d₆DMSO) 1.2 (s, 8H), 2.9 (s, 4H), 4.15 (s, 4H), 5.9 (t, 2H), 6.5 (d, 2H),6.8 (d, 2H), 7.9 (t, 2H), 8.7 (s, 2H).

EXAMPLE 5 Preparation of1,8-di-(3-hydroxy-2-oxopyrid-1-yl)-acetamido)-octane

3-Benzyloxy-1-succinimyloxycarbonylmethylpyrid-2-one--prepared asdescribed in Example 4--is reacted with 1,8-diamino-octane in ananalogous manner to that described for the reaction of this compoundwith 1,6-diaminohexane in Example 4 to yield the title compound in 76%yield, m.p. 238°-239° C.; ν_(max) (nujol) 1650, 1580, 1560 cm⁻¹ ; δ(d₆DMSO) 1.2 (s, 12H), 4.4 (s, 4H), 5.9 (t, 2H), 6.6 (d, 2H), 6.9 (d, 2H),8.0 (t, 2H), 8.8 (s, 2H).

EXAMPLE 6 Preparation ofN-[2-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)ethyl]-3[3-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)]-propylamineand other related compounds

3-Benzyloxy-2-methyl-4-pyrone (5.2 g, crude) is dissolved in a mixtureof ethanol (100 ml) and water (200 ml) containing sodium hydroxide (2g), the pH of the resulting solution being about 12 to 13. Thetri-amine, N-(2-aminoethyl)-1,3-propanediamine (0.75 g), is then addedto give a reaction mixture which contains a 3.75 molar excess of the4-pyrone to tri-amine. The mixture is stirred at room temperature for 6days and is then acidified with concentrated HCl to pH 2 and rotaryevaporated to give a brown solid. This solid is extracted with ethanolfrom which a white solid is obtained on rotary evaporation.Recrystalliation from water givesN-[2-(3-hydroxy-2-methyl-4-oxopyrid-1-yl]-3-(3-hydroxy-2-methyl-4-oxopyrid-1-yl-propylamine(1.5 g). This product is hydrolyzed by refluxing in 40% w/w HBr in CH₃CO₂ H (ca 300 ml) on a steam bath for 30 minutes. The acids are removedby rotary evaporation (<1 mm Hg) and water is added to the resulting oilto give a white precipitate. This precipitate is redissolved by heatingand recrystallised by cooling to give the title compound.sup.(1) (0.66g, 31%) as large elongated off-white crystalls of m.p. 137°-140° C.; γ_(max) (nujol) 840, 1020, 1500, 1540, 1630 and 3400 cm⁻¹ ; δ(d₆ DMSO)2.2 (m, 2H), 2.5 (s, 3H), 3.1 (m, 2H), 3.5 (m,2H), 4.4-4.8 (m, 4H), 7.2(d, 1H)) and 8.4 (d, 1H).

(A) N-[2-(Aminoacetamido)-ethyl]-3-(aminoacetamido)-propylamine

The p-nitrophenyl ester of N-benzylglycine (2 g) andN-(2-aminoethyl)-1,3-propanediamine (0.18 g) are dissolved inethylacetate (30 ml). After standing for 1 hour, the mixture isfiltered, evaporated and resulting solid triturated with diethyl ether.Recrystallisation of the product from ethanol yieldsN-[2-(benzylaminoacetamido)-ethyl]-3-(benzylaminoacetamido)-propylamineas a solid, ν_(max) (nujol) 1680, 1630, 1540 cm⁻¹ ; δ(d₆ DMSO) 1.5(quintet, 2H), 2.5 (m, 4H), 3.1 (q, 4H), 3.6 (d, 4H), 5.0 (s, 4H), 7.3(s, 10H), 7.4 (m, 2H), 7.7 (m, 2H).

Hydrogenation of this compound in ethanol containing 1% v/v of aceticacid over palladium/charcoal gives a 75% yield of the title compound asa viscous oil.

(B)N-(Aminoacetamido)-N-[2-(aminoacetamido)-ethyl]-3-(aminoacetamido)-propylamine

The p-nitrophenyl ester of N-benzylglycine andN-(2-aminoethyl)-1,3-propane diamine are reacted in the same proportionsas in (A) above but in dimethyl formamide as the solvent to yield, afterworking up in a similar manner,N-(N-benzylglycyl)-N-[2-(benzylaminoacetamido)-ethyl]-3-(benzylaminoacetamido)-propylaminein 65% yield, δ(d₆ DMSO) 1.6 (m, 2H), 3.2 (m, 6H), 3.5 (d, 4H), 3.8 (d,2H), 5.0 (s, 6H), 7.3 (s, 15H), 7.4 (m, 3H), 7.8 (m, 3H). Hydrogenationas in (A) above yields the title compound in 75% yield as an oil.

(C)N-(Aminoacetamido)-N-[3-(aminoacetamido)-propyl]-4-(aminoacetamido)-butylamine

The p-nitrophenyl ester of N-benzylglycine andN-(3-aminopropyl)-1,4-butane diamine are reacted together and thereaction mixture worked up in a similar manner to that described in (B)above for the reaction of this ester withN-(2-aminoethyl)-1,3-propanediamine to giveN-(N-benzylglycyl)-N-[3(benzylaminoacetamido)-propyl]-4-(benzylaminoacetamido)-butylamine,ν_(max) (nujol) 1660, 1625 cm⁻¹ ; δ(d₆ DMSO) 1.4 (m, 6H), 3.1 (m, 6H),3.2 (s, 4H), 3.5 (d, 4H), 3.8 (d, 2H), 5.0 (s, 6H), 7.3 (s, 15H), 7.4(m, 3H), 7.8 (m, 3H). Hydrogenation as in (A) above yields the titlecompound in 70% yield as an oil.

The three amines prepared as described under (A), (B) and (C) arereacted with 3-benzyloxy-2-methyl-4-pyrone in an analogous manner tothat described in this Example for N-(2-aminoethyl)-1,3-propanediamineto give, respectively,N-[2-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-ethyl]-3-(3-hydroxy-2-methyl-4oxopyrid-1-ylacetamido)-propylamine,N-(3-hyxdroxy-2-methyl-4-oxopyrid-1-ylacetyl)-N-[2-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)ethyl]-3-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-propylamineandN-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetyl)-N-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-propyl]-4-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-butylamine.

EXAMPLE 7 Preparation ofN-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-ylpropionamido)-propyl]-4-(3-hydroxy-2-methyl-4-oxopyrid-1-ylpropionamido)-butylamine

3-Benzyloxy-1-(2'succinimyloxycarbonylethyl)-2-methylpyrid-4-one isprepared from 3-benzyloxy-1-carboxyethylpyrid-2-one by reaction withN-hydroxysuccinimide as described in Example 1, the compound beingisolated in an analogous manner to that described in Example 4 for thecorresponding 1-(2'succinimyloxycarbonylethyl) compound. The compound isobtained in 60% yield as a solid of m.p. 144146° C.; ν_(max) (nujol)1800, 1770, 1735, 1625, 1570 cm⁻¹ ; δ(CDCl₃) 2.0 (s, 3H), 2.8 (s, 4H),2.9 (t, 2H), 4.1 (t, 2H), 5.1 (s, 2H), 6.3 (d, 1H), 7.3 (s, 5H), 7.3 (d,1H).

3-Benzyloxy-1-(2'succinimyloxycarbonylethyl)-2-methylpyrid-4-one (0.5 g)is dissolved in methylene chloride (30 ml) and the solution is addeddropwise over 15 minutes to spermidine [N-(3-aminopropyl-butylamine)(0.19 g) in methylene chloride (10 ml). The resulting solution, isallowed to stand for 1 hour and is then washed with water, dried (Na₂SO₄) and evaporated to giveN-[3-(3-benzyloxy-2-methyl-4-oxopyrid-1-ylacetamido)-propyl]-4-(3-benzyloxy-2-methyl-4-oxopyrid-1-ylacetamido)-butylaminein 70% yield as an oil, which on trituration with cold ethanol followedby recrystallisation from ethanol gives a white solid, m.p. 89° C.;ν_(max) (nujol) 3250, 1640, 1590 cm⁻¹ ; δ(d₆ DMSO) 1.4 (m, 6H), 2.4 (s,6H), 2.6 (m, 4H), 2.8 (m, 4H), 4.4 (t, 4H), 4.9 (s, 4H), 7.3 (s, 10 H),8.1 (m, 2H), 8.6 (d, 2H). Hydrogenation of this dibenzyl derivative overpalladium/charcoal in ethanol containing 1% v/v acetic acid in ananalogous fashion to that described in Example 4 yields the titlecompound as an oil.

EXAMPLE 8 Preparation ofN,N-di-[2-(3-hydroxy-2-oxopyrid-1-ylacetamido-ethyl]-2-(3-hydroxy-2-oxopyrid-1-ylacetamido)-ethylamine

3-Benzyloxy-1-succinimyloxycarbonylmethylpyrid-2-one--prepared asdescribed in Example 4--(2.1 g) is dissolved in dimethylformamide (50ml) and tri-(2'-aminoethyl)-amine (0.3 g) is added with stirring. Theresultant solution is stirred for 10 minutes and is then evaporated todryness. The residue is extracted with methylene chloride/water, theorganic layer being evaporated to dryness. The residue is dissolved in95% ethanol and hydrogenated over palladium/charcoal, filtration andevaporation yielding a solid which is disiccated over solid NaOH.Recrystallisation of the desiccated solid from 95% ethanol yields thetitle compound (0.82 g, 68%) as a white solid, m.p. 112°-114° C.;ν_(max) (nujol) 1650, 1590 cm⁻¹ ; δ(d₆ DMSO) 2.3 (s, 6H), 2.95 (s, 6H),4.4 (s, 6H), 5.9 (t, 3H), 6.55 (d, 3H), 6.9 (d, 3H), 8.0 (s, 3H).

EXAMPLE 9 The preparation ofN,N-di-(2-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)-propionamido]-ethyl)-2-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)-propionamido]-ethylamine

3-Benzyloxy-1-(2'-succinimyloxycarbonylethyl)-2-methylpyrid-4-one--preparedas described in Example 7--is reacted with tri(2'-aminoethyl)-amine inan analogous procedure to that described in Example 8 for the reactionof the corresponding 1-(2'-succinimylcarbonylmethyl) compound. Workingup in an analogous fashion givesN,N-di-(2-[3-(3-benzyloxy-2-methyl-4-oxopyrid-1-yl)-propionamido]-2-[3-(3-benzyloxy-2-methyl-4-oxopyrid-1-yl)propionamido]-ethylamine in 45% yield as a thick viscous oil, δ(CDCl₃)2.2 (s, 9H), 2.6 (t, 2H), 3.3 (m, 4H), 4.1 (t, 6H), 4.9 (s, 6H), 6.1 (d,3H), 7.3 (s, 15H), 7.6 (d, 3H), 8.8 (m, 3H). Hydrogenation of thistribenzyl derivative over palladium/charcoal in ethanol containing 1%v/v acetic acid in an analogous fashion to that described in Example 8yields the title compound.

EXAMPLE 10 The preparation ofN-(3-hydroxy-2-oxopyrid-1-ylacetyl)-N-[3-(3-hydroxy-2-oxopyrid-1-ylacetamido)-propyl]-3-(3-hydroxy-2-oxopyrid-1-ylacetamido)-propylamine

3-Benzyloxy-1-succinimylcarbonylmethylpyrid-2-one--prepared as describedin Example 4--(2 g) is dissolved in dimethylformamide (50 ml) and asolution of N-(3-aminopropyl)-1,3-diaminopropane in methanol (20 ml) isadded and the reaction mixture is allowed to stand overnight. Rotaryevaporation yields an orange oil which is extracted with methylenechloride/water. The organic layer is dried, evaporated to dryness anddesiccated over CaCl₂ to giveN-(3-benzyloxy-2-oxopyrid-1-ylacetyl)-N-[3-(3-benzyloxy-2-oxopyrid-1-ylacetamido)-propyl]-3-(3-benzyloxy-2-oxopyrid-1-ylacetamido)-propylamineas an oil in 60% yield; δ(d₆ DMSO) 1.6 (m, 4H), 3.0 (m, 8H), 4.3 (s,6H), 4.75 (s, 6H), 6.0 (t, 3H), 6.8 (d, 3H), 7.1 (d, 3H), 7.3 (s, 15H),8.3 (m, 2H). Hydrogenation of this tribenzyl derivative overpalladium/charcoal in ethanol containing 1% v/v acetic acid in ananalogous fashion to that described in Example 8 yields the titlecompound as an oil.

EXAMPLE 11 Partition data on linked 3-hydroxypyridones and their ironcomplexes

The partition coefficient K_(part), being the ratio (concentration ofcompound in n-octanol)/(concentraction of compound in aqueous phase) onpartition between n-octanol and aqueous tris hydrochloride (20 mM, pH7.4), is measured by spectrophotometry at 20° C. for various of thecompounds of the previous Examples and for their iron(III) complexes (at10⁻⁴ M). The solutions of the complexes are either produced, forcompound 8, by dissolving the pre-formed complex prepared as describedin Example 13, in the aqueous tris hydrochloride or, for compounds 5 and10, by formation in the buffer in situ by the admixture of a 3:2 or 1:1molar ratio, respectively, of the linked hydroxypyridone compound:ferricchloride, the pH thereafter being readjusted to 7.4 if necessary. Acidwashed glassware is used throughout and, following mixing of 5 ml of the10⁻⁴ M aqueous solution with 5 ml n-octanol for 1 minute, the aqueousn-octanol mixture is centrifuged at 1,000 g for 30 seconds. The tworesulting phases are separated for a concentration determination byspectrophotometry on each. For the metal-free linked hydroxypyridonecompounds, the range 220-340 nm is used for concentration determinationswhilst for the iron complexes, the range 340-640 nm is used.

Values typical of those obtained are shown in Table 2, the compoundsbeing identified by the same numbering system as that used in Table 1.

                  TABLE 2                                                         ______________________________________                                        Partition coefficients                                                                  Partition coefficient K.sub.part                                                            Iron complex                                          Compound    Free compound                                                                             [Fe.sup.III -(compound).sub.3 ]                       ______________________________________                                        5           0.017       0.10                                                  8           0.02        0.03                                                  10          0.2         0.3                                                   ______________________________________                                    

EXAMPLE 12 In vitro tests of iron binding capacity

The linked hydroxypyridone compounds used in this Example were preparedas described in various of the previous Examples and are identified inTables 3 and 4 below by the same numbering system as that used in Table1.

(1) Mobilisation of iron from ferritin

Horse spleen ferritin (Sigma) was used without further purification andits iron content was estimated spectrophotometrically at 420 nm. Theferritin solution in phosphate buffered saline (Dulbecco-OXOID, 10⁻⁶ M,pH 7.4) was incubated at 25° C. with the compound at 10⁻⁴ M for 6 and 24hours. Apoferritin (in admixture with ferritin) and the particularlinked hydroxypyridone Fe.sup.(III) complex were separated bychromatography on Sephadex G10. The absorption spectra of the high andlow molecular weight fractions were recorded and the percentage of ironremoved from the ferritin was calculated and is reported in Table 3. Forcomparative purposes, the procedure was repeated using a blank control,and also with the single ring, bidentate compound1-ethyl-3-hydroxypyrid-4-one (at 10⁻³ M). In addition, results reportedin the literature for similar tests with 1×10⁻³ M desferrioxamine(Crichton et al, J. Inorganic Biochem., 1980, 13, 305) and with 6×10⁻³ MLICAMS (Tufano et al, Biochem. Biophys. Acta, 1981, 668, 420 ) are alsogiven in the Table. It will be seen that the linked hydroxypyridonecompounds are less effective at removing iron from ferritin than thesingle ring compounds probably owing to the fact that the size of theiron complexes formed, like those of desferrioxamine and LICAMS, are toolarge to rapidly penetrate the ferritin pores.

                  TABLE 3                                                         ______________________________________                                        Removal of iron from ferritin                                                               Concen-     Percentage of                                                     tration     iron removed                                        Compound      M           6 hours 24 hours                                    ______________________________________                                        Control       --          <1      <1                                          1-ethyl-3-hydroxypyrid-                                                                     10.sup.-3   25      44                                          4-one                                                                         4             10.sup.-4   <1       3                                          8             10.sup.-4   3       10                                          Desferrioxamine                                                                             10.sup.-3   1.2     --                                          LICAMS        6 × 10.sup.-3                                                                       0       --                                          ______________________________________                                    

(2) Mobilisation of iron from transferrin

Human transferrin (Sigma) was loaded with iron(III) by the method ofBates and Schlaback, J. Biol. Chem. (1973) 248, 3228. ⁵⁹ Iron (III)transferrin (10⁻⁵ M) was incubated with a 4×10⁻³ M solution in tris HCl(0.1M, pH 7.4) of one of various pyridones as indicated in Table 4 forperiods of 6 hours. Apotransferrin (inadmixture with transferrin) andthe particular linked hydroxypyridone complex (Fe.sup.(III) complex wereseparated by chromatography or Sephadex G10. The 59^(Fe) content of bothfractions was determined and the percentage of iron removed fromtransferrin was calculated and is reported in Table 4. For comparativepurposes, this procedure was repeated with desferrioxamine and EDTA. Itwill be seen that three-ring compound 8, in particular, gives markedlybetter results than desferrioxamine or EDTA.

                  TABLE 4                                                         ______________________________________                                        Removal of iron from transferrin                                                            Percentage of iron removed                                      Compound      after 6 hours                                                   ______________________________________                                        5             32                                                              8             40                                                              Desferrioxamine                                                                             17                                                              EDTA          27                                                              ______________________________________                                    

EXAMPLE 13 Preparation of the iron complexes

The iron complex of 1,6-di-(3-hydroxy-2-oxypyrid-1-ylacetamido)-hexane(compound 4: described in Example 4) is prepared by either procedure (a)or procedure (b).

(a) An aqueous solution of 2 molar equivalents of ferric chloride isreacted for 5 minutes at room temperature with an aqueous solutioncontaining 3 molar equivalents of1,6-di-(3-hydroxy-2-oxypyrid-1-ylacetamido)-hexane.sup.(1). Theresultant solution is adjusted to pH 7.0 using 2 molar aqueous sodiumhydroxide and is then freeze dried. The resulting powder is extractedwith chloroform, filtered and the filtrate subjected to rotaryevaporation to give an essentially quantitative yield of a complexcontaining the 1,6-di-(3-hydroxy-2-oxopyrid-1-ylacetamido)-hexanebivalent anion and the ferric cation. Recrystallisation of the complexfrom ethanol gives wine red coloured crystals, m.p. 300° C., ν_(max)(nujol) 1,520, 1,610 cm⁻¹.

(b) An ethanolic solution of 2 molar equivalents of ferric chloride isreacted for 5 minutes at room temperature with a chloroform solutioncontaining 3 molar equivalents of1,6-di-(3-hydroxy-2-oxopyrid-1-ylacetamido)-hexane.sup.(1). Theresultant solution is neutralised by the addition of solid sodiumcarbonate, the precipitated sodium chloride removed by filtration andthe filtrate evaporated to give an essentially quantitative yield of acomplex containing the1,6-di-(3-hydroxy-2-oxopyrid-1-ylacetamido)-hexane bivalent anion andthe ferric cation, m.p. >300° C.

N,N-[2-(3-hydroxy-2-oxopyrid-1-ylacetamido)-ethyl]-2-(3-hydroxy-2-oxopyrid-1-ylacetamido)-ethylamine(compound 8: described in Example 8) is reacted with ferric chloride ina 1:1 molar ratio by either procedure (a) or (b) above to give theneutral iron(III) containing theN,N-[2-(3-hydroxy-2-oxopyrid-1-ylacetamido)-ethyl]-2-(3-hydroxy-2-oxopyrid-1-ylacetamido)-ethylaminetrivalent anion and the ferric cation in 1:1 preparation. This complexis obtained as wine red coloured crystals, m.p. >300° C., ν_(max)(nujol) 1530, 1580(w), 1620(w) and 1660 cm⁻¹.

EXAMPLE 14 In vitro tests on permeation of iron complexes into humanerythrocytes

The accumulation of iron by human erythrocytes which are associated withthe iron complexes of three compounds described in earlier Examples wasstudied by incubating for 1 hour at 37° C. a 5% suspension oferythrocytes in a medium consisting of the ⁵⁹ Fe labelled ironcomplex.sup.(1) (10⁻³ M) in aqueous sodium chloride (130 mM) buffered topH 7.4 by tris hydrochloride (2 ml). Following this period ofincubation, an aliquot of the erythrocyte/medium mixture was placedabove a layer of silicone oil and the erythrocytes separated bycentrifugation through the oil. The ⁵⁹ Fe levels associated with theerythrocytes and the incubation medium were then counted. By way ofcomparison, a similar experiment was concluded with ferric citrate. Theresults obtained are shown in Table 5 where the amount of the complexentering erythrocytes (n.mole) is given, the quoted values being in eachcase the mean of at least three determinations. It will be seen thateach of the iron complexes compares well with ferric citrate.

.sup.(1) The iron complexes of the compounds are prepared in situ in thebuffered solution using the compound and Fe⁵⁹ Cl₃ in exactly similarratios to those described in Example 13 for compounds 4 and 8, and forcompound 5 in the same ratio as described in that Example for compound4.

                  TABLE 5                                                         ______________________________________                                        Uptake of complexes by erythrocytes                                                           Amount of complex                                             Compound        entering erythrocytes (n.mole)                                ______________________________________                                        Fe.sup.III complex of compound 4                                                              52                                                            Fe.sup.III complex of compound 5                                                              85                                                            Fe.sup.III complex of compound 8                                                              45                                                            Fe.sup.III citrate                                                                            <5                                                            ______________________________________                                         EXAMPLE 15

In vitro tests on permeation of rat jejunal sac by iron complexes

The iron uptake into the serosal space of the inverted rat jejunal sacwas compared for the iron complexes of two compounds described inearlier Examples. Rats (male Sprague Dawley, 60 g) were killed and thejejunum removed, everted and cut into three segments (4 cm length). Thesegments were tied at both ends and filled with Krebs Ringer buffer (0.2ml) and incubated in Krebs Ringer buffer containing ⁵⁹ Fe complexes at37° C. for periods up to 1 hour (the iron complexes were prepared insitu by an analogous procedure to that described in Example 14). Thecontents of the sac were counted for ⁵⁹ Fe and measuredspectrophotometrically.

The results obtained for the two iron complexes according to the presentinvention and, by way of comparison, for ferric citrate (this being oniron compounds which is contained in preparations marketed for thetreatment of iron deficiency anaemia) are shown in Table 6, the ironuptake for each compound being shown relative to that for ferricchloride as 1. It will be seen that the complexes of the presentinvention each provide a level of iron uptake which is significantlyhigher than the level observed for ferric citrate.

                  TABLE 6                                                         ______________________________________                                                            Relative                                                                      Iron                                                      Compound            Uptake                                                    ______________________________________                                        FeCl.sub.3           1                                                        Fe.sup.III complex of compound 5                                                                  15                                                        Fe.sup.III complex of compound 8                                                                  11                                                        Fe.sup.III nitrate   2                                                        ______________________________________                                    

We claim:
 1. An iron complex of a compound containing 2 to 100 ringscarrying adjacent hydroxy and oxo groups, said rings being selected from3-hydroxypyrid-2-ones, 3-hydroxypyrid-4-ones and 1-hydroxypyrid-2-onesand being covalently linked to each other through linking groupscontaining between 6 and 24 carbon atoms and which are either wholly ofa hydrocarbon nature or which additionally contain one or more groups,which may be the same or different, selected from --O--, --S--, --NH--,##STR35## --CONH-- and ##STR36##
 2. The complex according to claim 1, inwhich the linked rings are substituted or unsubstituted3-hydroxypyrid-2-ones and/or 3-hydroxypyrid-4-ones.
 3. The complexaccording to claim 2, in which the rings are either unsubstituted apartfrom linking groups or are additionally substituted only on one or morering carbon atoms by an aliphatic hydrocarbon group of 1 to 6 carbonatoms.
 4. The complex according to claim 3, in which the rings areeither unsubstituted apart from linking groups or are additionallysubstituted only on one ring carbon atom.
 5. The complex according toclaim 3, in which the rings are 2-methyl-3-hydroxypyrid-4-one orunsubstituted 3-hydroxypyrid-2-one.
 6. The complex according to claim 1,in which two, three or four of said rings are linked.
 7. The complexaccording to claim 6, in which three of said rings are linked.
 8. Thecomplex according to claim 1, in which two 3-hydroxypyrid-2-one or two3-hydroxypyrid-4-one rings are linked through the nitrogen atoms thereofby a group --(CH₂)_(n) --, --(CH₂)_(a) --NH--(CH₂)_(b) --, --(CH₂)_(c)--CONH--(CH₂)_(d) --NHCO--(CH₂)_(c) -- or --(CH₂)_(c) --CONH--(CH₂)_(e)--NH--(CH₂)_(f) --NHCO--(CH₂)_(c) --, wherein n is an integer from 6 to12, a and b are each separately an integer from 2 to 6, c is an integerfrom 1 to 5, d is an integer from 2 to 8, and e and f are eachseparately an integer from 2 to
 4. 9. The complex according to claim 8,in which c is an integer from 1 to 3 and the size of the third andfourth types of linking group is limited by the requirement that 4≦2c+d≦12 and 6≦2 c+e+f≦12, respectively.
 10. The complex according toclaim 8, in which n is 8 to 10; a and b are each separately 2 to 5; c is1 and d is 6, or c is 2 and d is 2 or 4; and c is 1, e is 2 and f is 3or c is 2, e is 2 and f is
 2. 11. The complex according to claim 1, inwhich three 3-hydroxypyrid-2-one or three 3-hydroxypyrid-4-one rings arelinked through the nitrogen atoms thereof by a group ##STR37## wherein cis an integer from 1 to 5, e and f are each separately an integer from 2to 4, g is an integer from 1 to 3 and h is an integer from 2 to
 4. 12.The complex according to claim 11, in which c is an integer from 1 to 3and the size of the first linking group is limited by the requirementthat 6≦2c+e+f≦12 whilst the size of the second linking group is limitedby the requirement that for each of 2c+e+f, 2c+e+h and 2c+f+h the sum isgreater than or equal to 6 but less than or equal to
 12. 13. The complexaccording to claim 12, in which c is 1, e is 2, f is 3 and g is 1, or cis 1, e is 3, f is 4 and g is 1, or c is 1, e is 3, f is 4 and g is 2;and c=1 and e=f=h=2, or c=2 and e=f=h=2.
 14. The complex according toclaim 1 being a neutral iron(III) complex.
 15. The complex according toclaim 14, in which the proportion of linked rings:ferric cations is 3:1.16. The complex according to claim 7 being a neutral 1:1compound:iron(III) complex.
 17. The complex according to claim 11 beinga neutral 1:1 compound:iron(III) complex.
 18. The complex according toclaim 1, being the neutral 1:1 compound:iron(III) complex of a compoundselected from the group consisting ofN-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetyl)-N-[2-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-ethyl]-3-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-propylamine,N-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetyl)-N-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-propyl]-4-(3-hydroxy-2-methyl-4-oxopyrid-1-ylacetamido)-butylamine,N,N-di-(2-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)-propionamido]-ethyl)-2-[3-(3-hydroxy-2-methyl-4-oxopyrid-1-yl)-propionamido]-ethylamine,N,N-di-[2-(3-hydroxy-2-oxopyrid-1-ylacetamido)-ethyl]-2-(3-hydroxy-2-oxopyrid-1-ylacetamido)-ethylamineandN-(3-hydroxy-2-oxopyrid-1-ylacetyl)-N-[3-(3-hydroxy-2-oxopyrid-1-ylacetamido)-propyl]-3-(3-hydroxy-2-oxopyrid-1-ylacetamido)-propylamine.19. A pharmaceutical composition comprising a complex according to claim1, together with a physiologically acceptable diluent or carrier.
 20. Apharmaceutical composition comprising a complex according to claim 16,together with a physiologically acceptable diluent or carrier.
 21. Apharmaceutical composition comprising a complex according to claim 16and an iron chelating agent, together with a physiologically acceptablediluent or carrier.
 22. The pharmaceutical composition according toclaim 21, in which the iron chelating agent is a compound containing twoor more rings carrying adjacent hydroxy and oxo groups, said rings beingselected from 3-hydroxypyrid-2-ones, 3-hydroxypyrid-4-ones and1-hydroxypyrid-2-ones and being covalently linked to each other throughlinking groups which are either wholly of a hydrocarbon nature or whichadditionally contain one or more groups, which may be the same ordifferent, selected from --O--, --S--, ##STR38## --CONH-- and ##STR39##23. The pharmaceutical composition according to claim 22, in which theiron chelating agent is the same compound as in the iron complex, or aphysiologically acceptable salt thereof, in uncomplexed form.
 24. Thecomplex of claim 1, wherein said linking groups are either (i) wholly ofan aliphatic hydrocarbon nature or (ii) of an aliphatic hydrocarbonnature and additionally containing one or more groups, which may be thesame or different, selected from --NH--, ##STR40## --CONH--, and##STR41##
 25. The complex of claim 1, in which two or three3-hydroxypyrid-2-one rings are linked through the nitrogen atom thereof.26. The complex of claim 25, in which the nitrogen atoms of the ringsare separated by 6 to 12 atoms.
 27. The complex of claim 25, in whichthe nitrogen atoms of the rings are separated by 8 to 10 atoms.